CN112280693A

CN112280693A - Aspergillus strain GW13 and application thereof

Info

Publication number: CN112280693A
Application number: CN202011332090.6A
Authority: CN
Inventors: 宋福行; 李二伟; 徐秀丽; 王龙; 杨娜; 李红花; 张楷
Original assignee: Beijing Technology and Business University
Current assignee: Beijing Technology and Business University
Priority date: 2020-11-24
Filing date: 2020-11-24
Publication date: 2021-01-29

Abstract

The invention discloses an Aspergillus sp strain GW13 CGMCC No.19931 and application thereof in preparation of variegated aflatoxin. The strain has been preserved in China general microbiological culture Collection center (CGMCC for short, No. 3 of Xilu No.1 of Beijing, Chaoyang, Kyoho) as a patent strain in 16.7.2020, and registration number of the preservation center is CGMCC No. 19931. The invention effectively solves the technical problem of preparing the high-purity aflatoxin standard substance in the industry.

Description

Aspergillus strain GW13 and application thereof

Technical Field

The invention relates to the technical field of producing variegated aflatoxins by aspergillus.

Background

The variegated aspergillotoxin (ST) is mainly produced by variegated aspergilli, aspergillus pyronarius, aspergillus discolourus, aspergillus unguis, chaetomium, aspergillus flavus and the like. The mold is widely present in grains such as wheat, barley, corn, peanut, soybean and the like, and the aspergillus versicolor is the most toxic.

The aflatoxins are among the carcinogenic mycotoxins. Although the toxicity of the product is weaker than that of aflatoxin, the main toxigenic bacteria such as aspergillus versicolor and aspergillus nidulans are widely distributed in food and feed, and the proportion of toxigenic bacteria is high, so that the harm to people, livestock and poultry is not negligible.

Mycotoxin is a necessary inspection item in the production process of food, and commonly used mycotoxin detection methods comprise a performance liquid chromatography, a gas chromatography, enzyme-linked immunosorbent assay and a plurality of chromatography-mass spectrometry coupling technologies, but most of the detection methods rely on imported standard substances as reference substances at present, and domestic production cannot be realized.

How to prepare high-purity aflatoxin standard products is a technical problem which is not solved in the field.

Disclosure of Invention

The first purpose of the invention is to provide an Aspergillus sp strain GW13 belonging to Aspergillus sp, which has been deposited in China general microbiological culture Collection center (CGMCC for short, address: Beijing city Shangyang district North Cheng Xilu No.1 hospital 3) as a patent strain at 2020, 7, 16 and the registration number CGMCC No. 19931.

The second purpose of the invention is to provide the application of the strain in preparing the aflatoxin by fermentation and purification.

Further, the structural formula of the aflatoxin is shown in a formula I:

the invention effectively solves the technical problem of preparing the high-purity aflatoxin standard substance in the industry.

Drawings

FIG. 1 is a drawing of a compound of formula I¹H nuclear magnetic resonance spectrum.

FIG. 2 is a drawing of a compound of formula I¹³C nuclear magnetic resonance spectrum.

Detailed Description

Example 1

1. Collection of Strain GW13

A strain is separated from corns collected in Shaanxi province Yanyang city in 9 months in 2019 and is named as strain GW 13.

2. Identification of Strain GW13

The BT partial sequence of the strain GW13 is shown in a sequence 1(SEQ ID NO.1) of a sequence table, and has the highest sequence similarity with GENBANK ACCESSION NO. JN854007.1, wherein the similarity is 99.5%.

According to the above identification results, the strain GW13 belongs to Aspergillus sp.

3. Collection of Strain GW13

Strain GW13, belonging to Aspergillus spYear 2020, 7 and 16Is preserved in China general microbiological culture Collection center (CGMCC for short, with the address of No. 3 Xilu 1. of Beijing Kogyo-Yang) with the registration number of CGMCCNo.19931。

4. Preparation of variegated aspergillotoxin by strain GW13

1) Inoculating strain GW13 preserved in-80 deg.C glycerol tube onto potato glucose agar (PDA) culture medium, and standing at 28 deg.C for 5-10 days. The PDA culture medium is as follows: 40g of Potato Dextrose Agar dry powder (Gibco, USA), distilled water to 1000mL, and sterilized at 115 ℃ for 30 min.

2) Washing the plate PDA culture medium in the step 1 with sterile water to collect fungal spore liquid, inoculating the collected spore liquid into a 1000mL triangular flask containing a fermentation culture medium, and performing static culture at 28 ℃ for 20 days to obtain a fermented product; the fermentation product consists of spores, hyphae and the solid culture medium.

In the step 2, the inoculation amount of the spore liquid on the PDA culture medium is preferably 5% of the weight of the fermentation culture medium (the culture medium consisting of rice, corn, wheat, brown rice and the like and water); in the culture medium consisting of rice, corn, wheat and water, the weight part ratio of the rice, the corn, the wheat and the water is 100 g: 100 mL;

the fermentation condition may be 20-35 deg.C, such as solid static culture at 28 deg.C for 7-35 days (specifically 20 days).

Fermentation medium: corn, rice, wheat, brown rice, etc., without being limited to these four media. Corn culture medium: 200g of rice was soaked in 200mL of water overnight and autoclaved at 121 ℃ for 20 min. Rice culture medium: soaking 200g corn grit in 200mL water overnight, and autoclaving at 121 deg.C for 20 min. Wheat culture medium: 200g of wheat was soaked in 200mL of water overnight and sterilized with high pressure steam at 121 ℃ for 20 min. Brown rice culture medium: soaking 200g brown rice in 200mL water overnight, and sterilizing with high pressure steam at 121 deg.C for 20 min.

3) 2.0L of an organic reagent (ethyl acetate: methanol 4:1) was added to the flask (containing the solid fermentation product) obtained in step 2, and the mixture was extracted at room temperature for 24 hours, and the supernatant was collected and extracted 3 times repeatedly, and the supernatants were combined. And distilling the supernatant by using a rotary evaporator under reduced pressure until the supernatant is dried to obtain a crude extract.

4) Dissolving the crude extract obtained in the step 3 by using a mixed solvent (1:1) of dichloromethane and methanol, and mixing the dissolved crude extract with silica gel (the mass ratio of the crude extract to the silica gel is 1: 2) after the solvent is volatilized, performing adsorption chromatography separation (the adsorption chromatography separation is thin-layer chromatography silica gel column separation, the model of the gel column is Sephadex LH-20), eluting twice by using dichloromethane, and then eluting five times by using a mixed solution of dichloromethane and methanol, wherein the volume ratio of the dichloromethane to the methanol is as follows in sequence: 99: 1. 99: 1. 98: 2. 98: 2 and 97: 3 successively 7 fractions were obtained.

5) And (3) sequentially obtaining 7 fractions from 7 fractions obtained in the step (4) by using a volume ratio of 2: 1, collecting 1 part of the mixture of dichloromethane and methanol as eluent for adsorption chromatography (gel column chromatography), sequentially obtaining 5 fractions, purifying the 5 th fraction (purifying by using a reverse phase chromatographic column, wherein the mobile phase is a methanol aqueous solution with the volume percentage concentration of 65 percent; the reverse phase chromatographic column can be an Agilent reverse phase chromatographic column with the model of RP-18, the column length of the chromatographic column is 250mm, the inner diameter of the chromatographic column is 9.4mm, and the detection wavelength is 254 nm), and obtaining the compound shown in the following formula I:

the numerical table is as follows: process for preparing compounds¹H NMR spectrum (see FIG. 1) and¹³c nuclear magnetic resonance spectrum (as in figure 2)

HPLC purity chromatogram of compound of formula I (fig. 3). The peak area analysis showed that the formula I content was 99.17%.

Sequence listing

<110> Beijing university of Industrial and commercial

<120> aspergillus strain GW-13 and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 404

<212> DNA

<213> Aspergillus (Aspergillus sp)

<400> 1

btgtgaccct tggcccagtt gttaccagca ccggactggc caaagacgaa gttgtcggga 60

cggaaaagct gaccgaaggg accggcacgg acagcgtcca tggtaccggg ctcgagatcg 120

acgaggacgg cacgaggaac gtacttgttg ccgctggcct cgttgaagta gacgttcata 180

cgctcgagct ggaggtcgga ggtaccattg taactgcatt atatcaggga atgtgttctg 240

tctcgtgttt tgatcgagtc ctggacgggt tgtactcaca caccggagcc atcgaggccg 300

tgctcaccgg agatggtctg cctatagagt cgttagcaaa aagcacgaaa ggagatacca 360

tctgaaatgg atgaaatttt cgacgcacca gaaagcagca ccga 404

Claims

1. Aspergillus sp strain GW13 CGMCC No. 19931.

2. Application of Aspergillus sp (GW 13) strain in preparation of aflatoxin.

3. Use according to claim 2, wherein the aflatoxin has the formula: